Array level Fourier ptychographic imaging

ABSTRACT

In one aspect an imaging system includes: an illumination system including an array of light sources; an optical system including one or more lens arrays, each of the lens arrays including an array of lenses, each of the lenses in each of the one or more lens arrays in alignment with a corresponding set of light sources of the array of light sources; an imaging system including an array of image sensors, each of the image sensors in alignment with a corresponding lens or set of lenses of the one or more lens arrays, each of the image sensors configured to acquire image data based on the light received from the corresponding lens or set of lenses; a plate receiver system capable of receiving a multi-well plate including an array of wells, the plate receiver system configured to align each of the wells with a corresponding one of the image sensors; and a controller configured to control the illumination of the light sources and the acquisition of image data by the image sensors, the controller further configured to perform: an image acquisition process including a plurality of scans, each scan associated with a unique pattern of illumination, each of the image sensors configured to generate an image for a respective one of the wells during each scan; and an image reconstruction process during which the controller performs a fourier ptychographic operation to generate a reconstructed image for each of the wells based on the image data captured for the respective well during each of the scans.

PRIORITY DATA

This application is a continuation of U.S. patent application Ser. No.15/636,494, titled “Array Level Fourier Ptychographic Imaging” by Kim etal. and filed on Jun. 28, 2017, which is a continuation of U.S. patentapplication Ser. No. 15/007,196 (issued as U.S. Pat. No. 9,829,695),titled “Array Level Fourier Ptychographic Imaging” by Kim et al. andfiled on Jan. 26, 2016, which claims benefit of and priority under 35U.S.C. 119(e) to U.S. Provisional Patent Application No. 62/107,628,titled “Development of 96-well Plate Fluorescence Imaging System” andfiled on Jan. 26, 2015, and to U.S. Provisional Patent Application No.62/107,631, titled “Real-time Cell Culture Monitoring via FourierPtychographic Microscopy” and filed on Jan. 26, 2015; each of which ishereby incorporated by reference in its entirety and for all purposes.This application is related to U.S. patent application Ser. No.15/007,159, filed on Jan. 26, 2016 and titled “MULTI-WELL FOURIERPTYCHOGRAPHIC AND FLUORESCENCE IMAGING,” which is hereby incorporated byreference in its entirety and for all purposes.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. OD007307awarded by the National Institutes of Health. The government has certainrights in the invention.

TECHNICAL FIELD

This disclosure relates generally to digital imaging, and morespecifically to Fourier ptychographic (FP)-based techniques for imagingan array of sample wells in parallel.

BACKGROUND

Multi-well plate readers are key pieces of bioscience equipment used toquickly obtain fluorescence and absorbance information from samples suchas live cultures grown in multi-well plates (for example, 96 wellplates). A typical reader takes 10 seconds to acquire a complete set offluorescence or absorbance measurements. However, conventional platereaders do not provide any image information. This represents asignificant loss or discarding of image information. For example,imaging of samples including live tissue cultures can reveal cellstructure and health information that can provide a wealth of insight tothe user. For example, image information of a well that returns anegative fluorescence signal in a toxicity screen can quickly inform auser as to whether the negative signal is due to the cell death,compromised growth, contamination, or other reasons. Generally, tocollect image information, the multi-well plate would have to be putinto a second sophisticated system that uses a microscope to slowly scanand image each well of the plate individually on a sequential basis.Because such conventional techniques are based on a singular microscope,the process is very slow. The complete process can take upwards ofapproximately 150 minutes or more for an entire multi-well plate. Such asignificant amount of machine time is an inefficient and prohibitive ifnumerous multi-well plates are to be imaged, for example, because suchlatency can compromise the time schedule of the experiment design. Inview of these constraints, it is not surprising that users often onlytake this extra imaging measurement step for a small fraction of thesamples, or when situations absolutely demand imaging.

SUMMARY

Certain aspects of this disclosure pertain to Fourier ptychographicimaging systems and methods.

In one aspect an imaging system includes: an illumination systemincluding an array of light sources; an optical system including one ormore lens arrays, each of the lens arrays including an array of lenses,each of the lenses in each of the one or more lens arrays in alignmentwith a corresponding set of light sources of the array of light sources;an imaging system including an array of image sensors, each of the imagesensors in alignment with a corresponding lens or set of lenses of theone or more lens arrays, each of the image sensors configured to acquireimage data based on the light received from the corresponding lens orset of lenses; a plate receiver system capable of receiving a multi-wellplate including an array of wells, the plate receiver system configuredto align each of the wells with a corresponding one of the imagesensors; and a controller configured to control the illumination of thelight sources and the acquisition of image data by the image sensors,the controller further configured to perform: an image acquisitionprocess including a plurality of scans, each scan associated with aunique pattern of illumination, each of the image sensors configured togenerate an image for a respective one of the wells during each scan;and an image reconstruction process during which the controller performsa fourier ptychographic operation to generate a reconstructed image foreach of the wells based on the image data captured for the respectivewell during each of the scans.

In another aspect an imaging method performed by an imaging system isdescribed, the imaging system including an illumination system includingan array of light sources; an optical system including one or more lensarrays, each of the lens arrays including an array of lenses, each ofthe lenses in each of the one or more lens arrays in alignment with acorresponding set of light sources of the array of light sources; animaging system including an array of image sensors, each of the imagesensors in alignment with a corresponding lens or set of lenses of theone or more lens arrays, each of the image sensors configured to acquireimage data based on the light received from the corresponding lens orset of lenses; a plate receiver system capable of receiving a multi-wellplate including an array of wells, the plate receiver system configuredto align each of the wells with a corresponding one of the imagesensors; and a controller configured to control the illumination of thelight sources and the acquisition of image data by the image sensors,the method comprising: performing an image acquisition process includinga plurality of scans, each scan associated with a unique pattern ofillumination, each of the image sensors configured to generate an imagefor a respective one of the wells during each scan; and performing animage reconstruction process during which the controller performs afourier ptychographic operation to generate a reconstructed image foreach of the wells based on the image data captured for the respectivewell during each of the scans.

These and other features are described in more detail below withreference to the associated drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a block diagram of an example imaging system capable ofFourier ptychographic (FP) imaging according to some implementations.

FIG. 2A shows a schematic diagram of an example imaging system capableof FP imaging according to some implementations.

FIG. 2B shows a cross-sectional perspective view of the imaging systemof FIG. 2A.

FIG. 3A shows a top view of an example sample platform having amulti-well plate positioned thereon according to some implementations.

FIG. 3B shows a bottom view of an example illumination system accordingto some implementations.

FIG. 3C shows a top view of an example lens array according to someimplementations.

FIG. 3D shows a top view of an example image sensor system according tosome implementations.

FIG. 4A shows a diagram of a portion of an example optical arrangementincluding three lenses according to some implementations.

FIG. 4B shows a diagram of a portion of an example optical arrangementincluding four lenses according to some implementations.

FIG. 5 shows a flowchart illustrating an example FP imaging process forimaging a multi-well plate according to some implementations.

FIG. 6A shows a diagram of an example arrangement of light sources andwells illuminated according to a first illumination pattern during afirst scan according to some implementations.

FIG. 6B shows the arrangement of FIG. 6A illuminated according to a2^(nd) illumination pattern during a 2^(nd) scan.

FIG. 6C shows the arrangement of FIG. 6A illuminated according to a7^(th) illumination pattern during a 7^(th) scan.

FIG. 6D shows the arrangement of FIG. 6A illuminated according to an8^(th) illumination pattern during a 8^(th) scan.

FIG. 6E shows the arrangement of FIG. 6A illuminated according to a42^(nd) illumination pattern during a 42^(nd) scan.

FIG. 6F shows the arrangement of FIG. 6A illuminated according to a49^(th) illumination pattern during a 49^(th) scan.

FIG. 7 shows a flowchart of an example FP reconstruction processaccording to some implementations.

FIG. 8 shows a flowchart of another example FP reconstruction processaccording to some implementations.

FIG. 9 shows a flowchart depicting operations of an example calibrationprocess for determining the angles of incidence for each of the wells ofa multi-well plate.

FIG. 10A shows a vignette monochromic image captured during illuminationby a central LED of an LED matrix according to one example.

FIG. 10B is a converted black and white version of the color image ofFIG. 10A.

FIG. 10C shows an image captured during illumination by a center LED ofa color LED matrix according to another example.

FIG. 11 shows a lookup plot of LED displacement associated with x-shiftand y-shift of the center of the image with respect to the center of theimage sensor, according to an embodiment.

FIG. 12 shows a flowchart illustrating an example fluorescence imagingprocess 1200 for imaging a multi-well plate according to someimplementations.

DETAILED DESCRIPTION

The following description is directed to certain implementations for thepurposes of describing various aspects of this disclosure. However, aperson having ordinary skill in the art will readily recognize that theteachings herein can be applied in a multitude of different ways. Thus,the teachings are not intended to be limited to the implementationsdepicted solely in the Figures, but instead have wide applicability aswill be readily apparent to one having ordinary skill in the art.

As used herein, the conjunction “or” is intended herein in the inclusivesense where appropriate unless otherwise indicated; that is, the phrase“A, B or C” is intended to include the possibilities of A, B, C, A andB, B and C, A and C and A, B and C. Additionally, a phrase referring to“at least one of” a list of items refers to any combination of thoseitems, including single members. As an example, “at least one of: A, B,or C” is intended to cover: A, B, C, A-B, A-C, B-C, and A-B-C.

I. Introduction

Various aspects relate generally to imaging systems, devices, andmethods capable of use in Fourier ptychographic (FP) imaging, and morespecifically, to imaging systems, devices, and methods configured toenable FP imaging at an array level. For example, particularimplementations are directed to an imaging system configured to enablehigh resolution FP imaging of each well of a multi-well plate inparallel. Some implementations further relate to such an imaging systemfurther configured to perform fluorescence imaging of each well of themulti-well plate in parallel.

Traditionally, the resolution of an image sensor, such as a camerasystem, determines the fidelity of visual features in the resultantimages captured by the image sensor. However, the resolution of anyimage sensor is fundamentally limited by geometric aberrations in thelens or lenses used to focus light onto the image sensor. This isbecause the number of resolvable points for a lens, referred to as theSBP, is fundamentally limited by geometrical aberrations. While CMOS andCCD technologies have been demonstrated having imaging sensors withpixels in the 1 micron (μm) range, it remains a challenge to design andmanufacture lenses which have the resolving power to match theresolution of such image sensors. This problem is further exacerbated inimaging systems that are to be configured to scan multiple samples inparallel, for example, because the lenses are very limited in diameter.

FP refers generally to an imaging technique that enablesnon-interferometric phase imaging and near-wavelengthdiffraction-limited resolution. FP generally requires the collection ofmultiple scans of an object (for example, a sample in a well), each scanbeing acquired using light at a different illumination angle than theother scans. The light can be generated from coherent light sources suchas, for example, a light emitted diode (LED) array. The image datacaptured in the scans is then processed using a phase retrievalalgorithm enabling an iterative reconstruction of the object into ahigher resolution image. FP imaging is generally related to(conventional) ptychography in that it solves the phase problem bypermuting the role of the real and the Fourier space by swapping thefocusing element and the object. Among the advantages of FP imagingtechniques are the capabilities to use imaging optics with lowernumerical aperture, which increases the depth of focus, the workingdistance and the size of the field of view. FP imaging techniques alsoenable the correction of lens aberrations, leading to a much largerspace-bandwidth product (SBP) (the mathematical product of theresolution and the exploitable size of an image).

Some examples of microscope systems and methods using FP imagingtechniques are discussed in “Wide-field, high-resolution Fourierptychographic microscopy,” Nat. Photonics 7(9), 739-745 (2013), X. Ou,R. Horstmeyer, C. Yang, and G. Zheng, “Quantitative phase imaging viaFourier ptychographic microscopy,” Opt. Lett. 38(22), 4845-4848 (2013),R. Horstmeyer and C. Yang, “A phase space model of Fourier ptychographicmicroscopy,” Opt. Express 22(1), 338-358 (2014), X. Ou, G. Zheng, and C.Yang, “Embedded pupil function recovery for Fourier ptychographicmicroscopy,” Opt. Express 22(5), 4960-4972 (2014), X. Ou, R. Horstmeyer,G. Zheng, and C. Yang, “High numerical aperture Fourier ptychography:principle, implementation and characterization,” Opt. Express 23(3),3472-3491 (2015), J. Chung, X. Ou, R. P. Kulkarni, and C. Yang,“Counting White Blood Cells from a Blood Smear Using FourierPtychographic Microscopy,” PLoS One 10(7), e0133489 (2015), A. Williams,J. Chung, X. Ou, G. Zheng, S. Rawal, Z. Ao, R. Datar, C. Yang, and R.Cote, “Fourier ptychographic microscopy for filtration-based circulatingtumor cell enumeration and analysis,” J. Biomed. Opt. 19(6), 066007(2014), and R. Horstmeyer, X. Ou, G. Zheng, P. Willems, and C. Yang,“Digital pathology with Fourier ptychography,” Comput. Med. ImagingGraphics 42, 38-43 (2015), which are hereby incorporated by referencefor the discussion.

As introduced above, various aspects of this disclosure relate toimaging systems, devices and methods for implementing FP processingtechniques to obtain high-resolution images of an entire array ofsamples in parallel at the array level. To implement the FP techniques,each of the imaging systems described herein generally includes anillumination system, a sample loading system, an optical system and animaging system. The illumination system generally includes an array oflight sources, the optical system generally includes one or more arraysof lenses, and the imaging system generally includes an array of imagesensing devices. In some example implementations, the sample loadingsystem is configured to receive a multi-well plate including a pluralityof sample wells, each of which contains a sample of interest. Theimaging system can further include a controller for selectively turningon (or “powering,” “actuating” or “illuminating”) particular ones,subsets or patterns of the light sources to provide plane waveillumination of each of a plurality of the wells simultaneously during ascanning operation (“scan”). A plurality of scans are performed over thecourse of an entire image acquisition phase using different patterns ofillumination such that each of the wells is illuminated at a pluralityof incidence angles by the time the image acquisition phase is complete.

The lenses of the optical system focus light scattered or emitted by thesamples in response to the illumination onto corresponding imagesensors. Each image sensor is configured to capture a relativelylow-resolution image of a region of a corresponding one of the wellsbased on the light it receives from the respective lens or lenses of theoptical system. Over the course of the entire image acquisition phase,each image sensor generates a sequence of intensity distributionmeasurements (raw intensity images), one image being generated for eachof the scans. A processing device combines the relatively low-resolutionraw intensity images for each of the wells in the spatial frequencydomain using a Fourier ptychographic reconstruction process to correctaberrations and to render a single high-resolution image for each of thewells. In particular aspects, the processing device performs the Fourierptychographic reconstruction processing on each of the wellsindividually but in parallel with the processing of the image datacaptured from the other ones of the wells enabling the parallelgeneration of a high-resolution image for each of the wells concurrently(or “simultaneously”). The FP approach also enables digitally refocusingof the resultant reconstructed images, for example, even if the systemmisses the focal plane by as much as 0.3 mm or more. Digital refocusingis particularly useful as it simplifies the process of imaging—the wellplate does not need to be as precisely placed in order to get highresolution images.

II. Imaging System for Fourier Ptychographic (FP) Imaging andFluorescent Imaging

FIG. 1 shows a block diagram of an example imaging system 100 capable ofFourier ptychographic (FP) imaging according to some implementations. Ata high level, the imaging system 100 is configured or configurable toscan an array of samples at the array level; that is, to illuminate andcapture images of an entire array of samples in parallel. The imagingsystem 100 also can include parallel processing capabilities totransform and combine raw image data frames obtained for each of thesample wells to generate an FP-reconstructed image for each of thesample wells at the array level. The imaging system 100 includes anillumination system 102, a sample loading system 104, an optical system106 and an image sensor system 108. A controller 110 controls theoperations of the illumination system 102 and the image sensor system108. The controller 110 also is configured to receive the raw (orminimally pre-processed) image data from the image sensor system 108. Insome implementations, the controller 110 is further configured toexecute one or more algorithms on the raw image data to perform one ormore processing operations such as various FP imaging processesincluding aberration correction.

The illumination system 102 includes an array (or “matrix”) of lightsources. For example, each light source can include one or morelight-emitting diodes (LEDs). The controller 110 controls theillumination of the light sources, for example, by selectively poweringon or otherwise allowing only particular ones or subsets of the lightsources to form various illumination patterns at particular times andfor particular durations during various imaging scans. The opticalsystem 106 generally includes at least one array of lenses (referred tohereinafter as a “lens array”). Each lens array includes a plurality (or“multiplicity”) of lenses. The image sensor system 108 includes an arrayof image sensors, for example, an array of cameras or other suitableimaging devices. In various implementations, the arrangement and totalnumber T of lenses in each array matches the arrangement and totalnumber of image sensors in the imaging system as well as the arrangementand total number of wells in a multi-well plate to be imaged.

The sample loading system 104 is generally configured to receive asample array such as a conventional or commercially-available multi-wellplate (also referred to as a “well plate,” “microtiter plate,”“microplate,” or “microwell plate”). Each multi-well plate generallyincludes an array (typically a rectangular array) of wells arranged in aplurality of rows and a plurality of columns. In typical applications,samples are generally pipetted or otherwise deposited into the wells forimaging. In various implementations, the sample loading system 104 ismore specifically configured to receive a multi-well plate inserted orotherwise loaded into the sample loading system 104 such that the wells(for example, the bottom surfaces of the wells) of the multi-well plateare positioned along a particular plane between the light sources of theillumination system 102 and the lenses of the optical system 106. Thesample loading system 104 also functions to approximately align thecenters of the wells of the multi-well plate with the centers ofcorresponding lenses of the optical system 106 (although as will becomeclear below, precise alignment is not required for variousimplementations of the imaging system described herein).

During a scanning operation, light generated by the illumination system102 illuminates samples in the wells. In some imaging modes orprocesses, such as those for use in FP imaging or other bright-fieldimaging, the light incident on each sample is scattered by the physicalfeatures of the sample as it passes through the sample. In some otherimaging modes or processes, such as those for use in fluorescenceimaging, the light sources are configured to generate particularwavelengths of excitation light to excite fluorophores (for example,specialized proteins) in the sample. In such fluorescence imaging, theincident excitation light imparts energy into the fluorophores, whichthen emit light at lower energy wavelengths. A portion of the scatteredlight or emitted light then passes through the transparent bottom of thewell to a corresponding lens (or set of lenses) of the optical system106. The lens(es) below each respective well generally function to focusthe scattered or emitted light from the well onto a respective one ofthe image sensors of the image sensor system 108. Each image sensor isconfigured to capture the light and output a data signal including imagedata representative of the intensities of light received at particularlocations of the image sensor (referred to herein as a “light intensitydistribution,” “intensity distribution,” or simply as an “image” or“image frame”).

The image data output by each of the image sensors is then transmitted(or “sent” or “communicated”) to the controller 110. In someimplementations, the controller 110 is configured to process the rawimage data of each scan to generate processed image data. For example,in some implementations the controller 110 is configured or configurableby a user to perform one or more FP image processing operations on theraw image data. As described above, to generate an FP-reconstructedimage of each well in parallel, a plurality of scans are performed usingdifferent illumination patterns. The controller 110 interprets imagedata from the sequence of acquired intensity images, transforms therelatively low resolution image data frames associated with each of thescans into fourier space, combines the transformed raw image data,corrects for aberrations resulting from the lenses as well as the samplefeatures, and generates a single high resolution image for each of thesample wells. As described above, the imaging system 100 also can beconfigured to perform fluorescence imaging. As such, the controller 110can generally include functionality to interpret, process, and in someinstances combine fluorescence image data for each of the sample wellsin parallel.

To perform such parallel image processing, the controller 110 generallyincludes at least one processor (or “processing unit”). Exampleprocessors include, for example, one or more of a general purposeprocessor (CPU), an application-specific integrated circuit (ASIC), anprogrammable logic device (PLD) such as a field-programmable gate array(FPGA), or a System-on-Chip (SoC) that includes one or more of a CPU,ASIC, PLD as well as a memory and various interfaces. The controller 110also is in communication with at least one internal memory device 120.The internal memory device 120 can include a non-volatile memory arrayfor storing processor-executable code (or “instructions”) that isretrieved by the processor to perform various functions or operationsdescribed herein for carrying out various algorithms or other operationson the image data. The internal memory device 120 also can store rawand/or processed image data (including FP-reconstructed images). In someimplementations, the internal memory device 120 or a separate memorydevice can additionally or alternatively include a volatile memory arrayfor temporarily storing code to be executed as well as image data to beprocessed, stored, or displayed. In some implementations, the controller110 itself can include volatile and in some instances also non-volatilememory.

In some implementations, the controller 110 is configured orconfigurable by a user to output raw image data or processed image data(for example, after FP image processing) over a communication interface112 for display on a display 114. In some implementations, thecontroller 110 also can be configured or configurable by a user tooutput raw image data as well as processed image data (for example,after FP image processing) over a communication interface 116 to anexternal computing device or system 118. Indeed in some implementations,one or more of the FP imaging operations can be performed by such anexternal computing device 118. In some implementations, the controller110 also can be configured or configurable by a user to output raw imagedata as well as processed image data (for example, after FP imageprocessing) over a communication interface 122 for storage in anexternal memory device or system 124. In some implementations, thecontroller 110 also can be configured or configurable by a user tooutput raw image data as well as processed image data (for example,after FP image processing) over a network communication interface 126for communication over an external network 128 (for example, a wired orwireless network). The network communication interface 126 also can beused to receive information such as software or firmware updates orother data for download by the controller 110. In some implementations,the imaging system 100 further includes one or more other interfacessuch as, for example, various Universal Serial Bus (USB) interfaces orother communication interfaces. Such additional interfaces can be used,for example, to connect various peripherals and input/output (I/O)devices such as a wired keyboard or mouse or to connect a dongle for usein wirelessly connecting various wireless-enabled peripherals. Suchadditional interfaces also can include serial interfaces such as, forexample, an interface to connect to a ribbon cable. It should also beappreciated that one or more of the illumination system 102 and theimage sensor system 108 can be electrically coupled to communicate withthe controller over one or more of a variety of suitable interfaces andcables such as, for example, USB interfaces and cables, ribbon cables,Ethernet cables, among other suitable interfaces and cables.

The data signals output by the image sensors may in some implementationsbe mutliplexed, serialized or otherwise combined by a multiplexer,serializer or other electrical component of the image sensor systembefore being communicated to the controller 110. In suchimplementations, the controller 110 can further include a demultiplexer,deserializer or other device or component for separating the image datafrom each of the image sensors so that the image frames for each of thesample wells can be processed in parallel by the controller 110.

FIG. 2A shows a schematic diagram of an example imaging system 200capable of FP imaging according to some implementations. FIG. 2B shows across-sectional perspective view of the imaging system 200 of FIG. 2A.The imaging system 200 of FIG. 2 is an example of a physicalimplementation of the imaging system 100 of FIG. 1. The imaging system200 generally includes a housing or enclosure 202. In someimplementations, the enclosure 202 is formed of a metal, metallic alloyor plastic material. In some implementations, the enclosure 202 isformed of an optically opaque material and/or painted or otherwisecoated in an optically opaque layer to prevent (or “block” or “shield”)ambient or other externally-generated light from illuminating thesamples or the image sensors. This light shielding can be especiallyimportant in fluorescence imaging where the intensity of the emittedlight is relatively much lower than that of the excitation light anddecays rapidly.

In some implementations, the enclosure 202 surrounds a frame structure204. In the illustrated implementation, the frame structure 204 providesa rigid frame from which the various components of the imaging system200 can be supported. In some implementations, the frame structure 204is formed of a metal, metallic alloy or plastic material. In someimplementations, the frame structure 204 also is formed of an opticallyopaque material and/or painted or otherwise coated in an opticallyopaque layer. In some implementations, the enclosure 202 and the framestructure 204 are integrally formed together. In some otherimplementations, the enclosure 202 and the frame structure 204 areassembled together by screws, bolts, rivets, glue or other devices ormaterials so as to be rigidly fixed together. In the illustratedimplementation, the frame structure 204 includes alignment through-holes205 through which frame alignment rods 206 are passed and positioned. Insome implementations, the frame alignment rods 206 also are formed of ametal, metallic alloy or plastic material.

In some implementations, each of the illumination system, the sampleloading system, the optical system and the image sensor system arephysically supported by one or more of the enclosure 202, the framestructure 204 and the frame alignment rods 206 so as to be rigidly fixedin relative position and at particular distances from one another. Insome implementations, each of the illumination system, the sampleloading system, the optical system and the image sensor system includesone or more substrates having corresponding through-holes. For example,the illumination system can include a circuit board or other dielectricsubstrate 212. The array of light sources 213 (hidden from view in FIG.2A or 2B but indicated by an arrow as being under the circuit board 212)can be electrically and physically coupled onto or into the circuitboard 212. Conductive leads of the light sources 213 can be electricallycoupled with the controller 210 via conductive traces printed orotherwise deposited on a first or upper surface of the circuit board 212while the light-emitting portions of the light sources 213 can beoriented so as to radiate light away from a second or lower surface ofthe circuit board 212 toward the lenses of the optical system. In theillustrated implementation, a controller 210 (for example, implementingcontroller 110 of FIG. 1) is mounted on the same circuit board 212 asthe light sources 213. In some other implementations, the controller 210can be mounted onto a separate circuit board that is electricallycoupled with the circuit board 212 and thereby to the illuminationsystem.

As described above, the optical system can include one or more lensarrays, for example, 1, 2, 3, 4 or more lens arrays depending on theparticular application. In the illustrated implementation, the opticalsystem includes two lens arrays 216 ₁ or 216 ₂ each of which includes arespective substrate into which are formed, assembled or positioned anarray of lenses 217 ₁ or 217 ₂, respectively. The image sensor systemcan include a circuit board or other dielectric substrate 218. An arrayof image sensors 219 can be electrically and physically coupled onto orinto the circuit board 218. The active light-sensitive regions of theimage sensors 219 can be oriented away from a first or upper surface ofthe circuit board 218 toward the lenses of the optical system while theconductive leads of the image sensors 219 can be electrically coupledwith the controller 210 via conductive traces printed or otherwisedeposited on a second or lower surface of the circuit board 218 to acommunication interface (for example, a USB interface) that is thenconnected with the controller 210 via a cable.

In such an arrangement, each of the frame alignment rods 206 can passthrough corresponding through-holes in each of the substrates 212, 216and 218 during assembly to align the light sources and respective onesof the lenses and images sensors along a vertical direction (forexample, a z direction along a height of the imaging system 200). Morespecifically, the frame alignment rods 206 can ensure that each imagesensor 219 is aligned with a corresponding lens in each of one or morestacked lens arrays, and that each of the lenses in each lens array arealigned with one another and with a set of one or more light sources213. The enclosure 202 and/or frame structure 204 also can includeguides, ribs, shelves or other supported mechanisms extending alonginner surfaces of the enclosure or frame structure, respectively, tophysically support the respective substrates 212, 216 and 218 at theproper distances from one another along the vertical z direction. Suchan arrangement ensures that the light sources 213, lenses 217 and imagesensors 219 are suitably positioned relative to one another to properlyfocus light scattered or emitted by the samples in the wells 209 ontothe image sensors and, as described below, such that the angles ofincidence of the light generated by the light sources can be preciselydetermined or otherwise known.

As described above, the sample loading system is generally configured toreceive a sample array such as a conventional or commercially-availablemulti-well plate 208 including a rectangular array of wells 209 arrangedin a plurality of rows and a plurality of columns. In the illustratedimplementation, a sample array 208 can be loaded through an apertureslot 214 in the housing enclosure 202 and onto a sample platform 215 inthe sample loading system. The sample platform 215 also can includethrough-holes into which the frame alignment rods 206 can pass to ensurethat the sample platform 215 is aligned with the image sensor system,the optical system and the illumination system. Additionally, the sampleplatform 215 can include raised guides or ridges or other alignmentmechanisms to ensure that a loaded multi-well plate 208 is properlyoriented such the centers of each of the wells 209 are approximatelyaligned with the centers of the corresponding lenses 217 in the lensarrays 216 and with the centers of the image sensors 219. In someimplementations, the sample loading system further includes a door thatis coupled with the enclosure 202 or with the frame structure 204 via asliding mechanism or a hinge mechanism enabling the door to be openedand closed with ease to insert and remove multi-well plates 208. In someimplementations, the sample loading system can include a mechanical,electrical or electromechanical loading and ejecting mechanism thatautomatically pulls the multi-well plate 208 into the imaging system forimaging and that automatically ejects the multi-well plate when afterthe imaging has been performed. Such an automatic mechanism can betriggered electronically by a user via an input device (such as akeyboard or mouse), triggered by a button or touchscreen interface onthe enclosure, or automatically by the controller 210 when it detectsthat a plate is being loaded or when it determines that an imagingoperation is complete.

FIG. 3A shows a top view of an example sample platform 305 having amulti-well plate 308 positioned thereon according to someimplementations. The multi-well plate 308 includes a number T of samplewells arranged in a plurality of R rows and C columns. Examples ofcommercially-available multi-well plates include the Costar® well platesmanufactured by Corning®, the CELLSTAR® 96 W Microplate manufactured byGreiner, and the CytoOne® well plates. In the illustrated example, themulti-well plate includes 96 wells arranged in eight rows and twelvecolumns. In such an example, each of the wells can have a diameter ofapproximately 6 millimeters (mm). In some other implementations, theimaging system 200 can be configured to image other multi-well plateformats such as, for example, plates consisting of 6 wells, 12 wells, 96wells, 384 wells, 1536 wells, 3456 wells or 9600 wells. In someexamples, each well has a volume in the range of approximately 1nanoliter (nL) to approximately 100 milliliters (mL), and in some morespecific 96-well examples, a total volume in the range of approximately350 microliter (μL) to approximately 400 μL, and a typical workingvolume in the range of approximately 25 μL to approximately 340 μL.Although each well is typically of a cylindrical shape having a circularcross-section with an open top end and a closed bottom end, other shapescan be used, for example, square or other rectangular cross-sections areavailable. Each well is further defined by a bottom surface on which asample may be deposited. In some examples, the multi-well plate isformed from a plastic material, such as a thermoplastic material such aspolystyrene. In some examples, the multi-well plate 308 is formed from aglass material. In some examples, portions of the multi-well plateexcluding the bottom surfaces of the wells 309 can further includecarbon. For example, for fluorescent biologic assays it is generallydesirable that the sides of the wells 309 are black to absorb/blockexternal/ambient light while the bottoms of wells should beclear/transparent to light in the visual wavelengths and fluorescencewavelengths of interest. The sample platform 305 can either betransparent or include a cutout portion below the multi-well plate toenable light to pass from the samples to the lenses of the opticalsystem.

FIG. 3B shows a bottom view of an example illumination system accordingto some implementations. As described above, the illumination systemgenerally includes a printed circuit board or other dielectric substrate312 onto or into which array of light sources 322 can be electricallyand physically coupled. As described above, the light-emitting portionsof the light sources 322 are oriented so as to radiate light toward thelenses of the optical system (and consequently also the samples in thewells of the well plate). As an example, the array of light sources 322can be implemented with an LED matrix. Each light source 322 can includeone or more LEDs. For example, in some implementations, each lightsource 322 includes a set of three or more LEDs including a red LED, agreen LED and a blue LED. In some other implementations, such as thatillustrated in FIG. 3B, each light source 322 includes a single LED, andmore specifically, a single RGB (Red, Blue, Green) LED including a redsub-LED 324 a, a green sub-LED 324 b and a blue sub-LED 324 c. In otherwords, each RGB LED actually has 3 semiconductor light sources; one red,one blue and one green. Whether implemented as a set of three distinctred, green and blue LEDs or as a single RGB LED, each light source 322can be capable of producing any visible color of light by varying theintensities of the red, green and blue light (for example, in responseto particular voltage signals provided by the controller). Additionallyor alternatively, each light source 322 also can include an LED forgenerating light in non-visible parts of the spectrum, such as infraredlight or ultraviolet light.

In some implementations, each light source 322 occupies a footprint ofless than 1 mm by 1 mm. In implementations configured to image 96-wellplates, the center to center distance (or “pitch”) between each well canby 9 mm while the center to center distance between each light source322 can be 3 mm. This means that there will be room for three lightsources between the centers of adjacent neighboring wells. Thisarrangement and ratio of LEDs to wells ensures that multiple lightsources 322 can illuminate the samples—each at a different angle ofincidence. In some example implementations, the number L of distinctlight sources 322 that are desired to ensure a sufficient number n ofdifferent angles of incidence are obtained for each well can be foundaccording to equation 1 below.

$\begin{matrix}{L = {\lbrack {( {m*R} ) + {2( {\frac{( {\sqrt{n} - 1} )}{2} - \frac{m - 1}{2}} )}} \rbrack*{\quad\lbrack {( {m*C} ) + {2( {\frac{( {\sqrt{n} - 1} )}{2} - \frac{m - 1}{2}} )}} \rbrack}}} & (1)\end{matrix}$where n is the desired number of angles of incidence and m is a numberrepresentative of a scaling factor indicative of a ratio of the densityof light sources to the density of wells.

In the illustrated 96-well implementation, where the number of rows R ofwells is 8 and the number C of columns of wells is 12, and taking n tobe 49 and m to be 3, the number L of light sources 322 is 1120 (forexample, 1120 RGB LEDs arranged in 28 rows and 40 columns). In someimplementation, the illumination system can further include side-mountedlight sources (for example, high power LEDs, not shown) for use inincreasing the intensities of the excitation signals for fluorescenceimaging scans to, in turn, increase the intensities of the emissionsignals emitted by the fluorophores within the samples.

FIG. 3C shows a top view of an example lens array 316 according to someimplementations. Like the lens arrays of FIG. 2B, the lens array of FIG.3C includes a substrate including an array of lenses 326. As describedabove, the optical system includes at least one lens array 316, and invarious implementations, at least two (for example, 2, 3, 4 or more)lens arrays in a vertically-aligned (or “stacked”) arrangement. Asdescribed above, in multi-lens-array implementations, each of the lenses326 in each lens array 316 is in vertical alignment with a correspondingone of the lenses 326 in each of the other lens arrays 316. The numberof lens arrays 316 and the types of the lenses 326 in each of the lensarrays can be optimized for particular applications. Generally, each ofthe lenses 326 within a given lens array 316 will be identical to allthe other lenses within the array, and different than all of the otherlenses in the other lens arrays. In some implementations orapplications, the combined set of lens aligned and otherwise associatedwith each well can have a numerical aperture (NA) in the range ofapproximately 0.05 to approximately 0.6.

FIG. 4A shows a diagram of a portion of an example optical arrangement400 including three lenses according to some implementations. Generally,the arrangement 400 represents the lenses as aligned and positioned forone well of a multi-well plate. Thus, each of the wells of themulti-well plate would include an identical arrangement 400, with eachof the elements of each arrangement being provided at the array levelwith like elements. In the illustrated representation, the top line 402of the arrangement 400 represents the location of the sample within thewell (on or above the inner bottom surface of the well) while element404 represents the bottom of the well (between the inner bottom surfaceof the well and the exterior bottom surface of the well plate). Element406 a represents a first lens element of a first lens array, element 406b represents a second lens element of a second lens array, and element406 c represents a third lens element of a third lens array. The set oflens 406 a, 406 b and 406 c are configured to focus light onto an activesurface of a corresponding image sensor 408. The lines passing throughthe various lenses and other elements in FIG. 4A represent light raysoriginating from different regions of the sample. In the illustratedimplementation, the optical arrangement 400 further includes a portionof an optical filter 410, for example, for use in fluorescence imagingapplications to filter light at excitation signal wavelengths. Asdescribed above, the optical filter can in various implementationgenerally be positioned anywhere between the multi-well plate and theimaging system, including between various ones of the lens arrays.

FIG. 4B shows an enlarged view of a diagram of a portion of an exampleoptical arrangement 401 including four lenses according to someimplementations. The arrangement 410 is similar to the arrangement 400shown in FIG. 4A, for example, having a top line 412 representing thelocation of the sample and an element 414 representing the bottom of thewell. However, in the implementation of FIG. 4B, the optical arrangement410 includes four lenses: element 416 a representing a first lenselement of a first lens array, element 416 b representing a second lenselement of a second lens array, element 416 c representing a third lenselement of a third lens array and element 416 d representing a fourthlens element of a fourth lens array. The set of lens 416 a, 416 b, 416 cand 416 d are configured to focus light onto an active surface of acorresponding image sensor (not shown). In the illustratedimplementation, the optical arrangement 401 also includes a portion ofan optical filter 420, for example, for use in fluorescence imagingapplications to filter light at excitation signal wavelengths.

Again, the number and types of the lens arrays and corresponding lensescan generally be dependent on the application. As an example, in animplementation in which the imaging system can be used in GreenFluorescent Protein (GFP) imaging, an optical arrangement such as thatshown in FIG. 4B is well-suited, for example, providing better resultsthan a three-lens-array arrangement such as that shown in FIG. 4A.Generally, the lens characteristics of each lens may be particularlydesigned for different wavelengths.

Referring back to FIG. 3C, in some implementations, each lens array 316is formed of one integral piece of material. For example, the lens array316 can be formed through an injection molding or three-dimensional (3D)printing process. Traditionally, such lenses would not have sufficientlylow geometric aberrations to enable high resolution imaging of arelatively wide field of view at the scale need to image a large numberof wells simultaneously in parallel. However, the use of multiple lensarrays, and thus multiple lenses for each well, can at least partiallynegate the effects of geometrical aberrations. Generally, the morelenses that are used for each well, the more geometrical aberrations inthose lenses can be canceled out. Additionally, FP techniques asdescribed herein can be used to further correct for any aberrations orto remove other image artifacts enabling the reconstruction of a highresolution image.

In implementations designed for fluorescence imaging applications, theoptical system also includes an optical filter 220 located between thebottom surface of the multi-well plate 208 and the image sensor system.The optical filter 220 blocks excitation light from the illuminationsystem (as well as any other light not emitted by the fluorophores inthe samples within the multi-well plate) from striking the image sensors219 of the image sensor system. Continuing the example above, for GFPimaging, the optical filter should be a green filter, that is, a filterpassing wavelengths of light in a green portion of the visible spectrum.The excitation signal light should be higher energy light, and inparticular applications, blue light. This is because the greenfluorescent proteins absorb light in the blue portion of the spectrumand emit light in the green portion of the spectrum. The green filterthen enables the emitted light to pass through to the image sensorswhile blocking the excitation light. In some implementations, theexcitation light also can be turned off immediately before acquiring theimage data.

In some implementations or applications, the range of wavelengths of thebright field illumination for the FP imaging fall within the passband ofthe optical filter 220 so that the optical filter passes light from thelight sources 322 that passes through the samples in the wells. In suchinstances, the image sensors can acquire a sequence of uniquelyilluminated bright field images while leaving the filter 220 in place.Continuing with the example above, bright field FP imaging of the GFPsamples can be performed with green light. In some other instances, therange of wavelengths of the bright field illumination from the lightsources 322 do not fall within the passband of the optical filter 220.In other words, in instances in which it is necessary or desirable tokeep the optical filter within the imaging system during the FP imaging,the bright field FP imaging should be performed using light of the sameor similar color as the filter used in the fluorescent imaging, else thefilter should be removed during the FP image acquisition process. Insome implementations, the optical filter can be readily removable and/orreplaceable with one or more different optical filters capable offiltering and passing different wavelengths of light. For example, theoptical filter 220 can be inserted into another aperture slot in theenclosure 202. In such implementations, the bright field FPM imaging canbe performed with light of a different color or with white light.

In some implementations, the optical filter is fabricated from a glassor plastic material and is in the shape of a rectangular solid having awidth (along the x axis) and a length (along a y axis) sufficientlylarge to provide filtering for all of the light scattered or emitted bythe samples and incident on the lenses of the optical system. Insingle-channel fluorescence imaging applications, a single band or lowpass filter can be used; for multi-channel fluorescence imagingapplications, a multi band filter can be used. As described above,because the optical filter 220 does not affect the path of the light,the optical filter 220 can be positioned anywhere between the bottomsurface of the multi-well plate 208 and the image sensors 209 of theimage sensor system.

FIG. 3D shows a top view of an example image sensor system according tosome implementations. Like the image sensor system of FIG. 2B, the imagesensor system of FIG. 3D includes a circuit board or other dielectricsubstrate 318 onto which an array of T image sensors 328 arranged in Rrows and C columns can be electrically and physically coupled. In someimplementations, each of the image sensors 328 has a field of view (FOV)of the respective sample well in the range of 0.5 mm to 6 mm indiameter, and in one specification example implementation, an FOV of 1mm diameter. In some implementations, each of the image sensors 328 alsois capable of capturing raw images at a spatial resolution of 0.5 μm to5 μm (prior to subsequent FP processing). As described above, the activelight-sensitive regions of the image sensors 328 can be oriented awayfrom a first or upper surface of the circuit board 318 toward the lensesof the optical system while the conductive leads of the image sensors328 can be electrically coupled with the controller 210 via conductivetraces printed or otherwise deposited on a second or lower surface ofthe circuit board 318 and via a communication interface (for example, aUniversal Serial Bus (USB) interface) that is connected with thecontroller 210. In some implementations, each of the image sensors is anactive-pixel sensor (APS) device such as CMOS-based APS cameras.

In some implementations, the raw image data captured by each of theimage sensors is transferred to the controller 210 through a high speeddata transfer channel (for example, at a data rate greater than 5 Gb/s).In some implementations, the image sensor system further includes aliquid cooling system that circulates cooling liquid around the surfacessurrounding the image sensors.

III. Variable-Illumination Fourier Ptychographic Imaging Methods

As described above, the imaging systems 100 and 200 are capable of FPimage acquisition of each and all of the sample wells of an entiremulti-well plate in parallel. In particular implementations, the imagingsystems 100 and 200 also are capable of fluorescence image acquisitionof each and all of the sample wells of an entire multi-well plate inparallel. An image acquisition (sample) time refers to a time during theexposure duration of each of the image sensors during which each of theimage sensors measures a light intensity distribution to capture anintensity image for a respective well of the multi-well plate. The FPimaging process typically comprises a raw image acquisition (datacollection) phase (or “process”) and an FP reconstruction phase (or“process”). During the FP image acquisition process, the controllercauses the illumination system to turn on particular subsets or patternsof the light sources. For example, the FP image acquisition process caninclude a plurality of scanning operations (or “scans”), each of whichscans includes a respective image acquisition by each of the imagesensors in the image sensor system. Each of the scans is associated witha different pattern of illumination of the light sources. Throughout thecourse of the entire FP image acquisition process, each of the imagesensors in the array of image sensors acquires s intensity bright fieldimages (corresponding to s scans) while the light sources of theillumination system provide plane wave illumination of each of the wellsfrom n different (unique) respective illumination angles of incidence.During each scan, each image sensor acquires an image based on aparticular illumination pattern of the light sources. Generally, thenumber s of scans can be equal to the number n of unique illuminationincident angles desired for each well. In this way, assuming a fixedvalue of n, the number of scans is independent of the number T of wellsin the multi-well plate.

FIG. 5 shows a flowchart illustrating an example FP imaging process 500for imaging a multi-well plate according to some implementations. Forexample, the process 500 can be performed using the systems, devices andarrangements described above with respect to FIGS. 1-4. In someimplementations, the controller 110 is configured perform one or moreoperations of the FP imaging process 500. In some implementations, theFP process 500 begins in operation 502 with loading a multi-well plateinto the imaging system. In operation 504, the controller initializesthe illumination system and the image sensor system. Initializing theillumination system in operation 504 can include retrieving illuminationpattern information from a non-volatile memory and loading the retrievedillumination pattern information into a volatile memory for subsequentuse in performing a series of sequential scanning operations.Initializing the image sensor system in operation 504 can includepowering on or otherwise preparing the image sensors to receive lightand to generate image data. In some implementations, initializing theillumination system and the image sensor system in operation 504 alsocan include a calibration operation to determine the actual values ofthe angles of incidence that will illuminate each of the wells.

After initialization, the controller performs an s^(th) scan (where s isan integer between 1 and n, inclusive, and where n is the number ofangles of incidence). As described above, during each scan, thecontroller causes the illumination system to produce a unique pattern ofillumination and causes the image sensor system to capture/acquire animage for each of the wells. In some implementations, each scan can beperformed or conceptualized as a sequence of sub-operations. Forexample, each scan can include a sequence of operations 506, 508, 510(and in some cases 512). In block 506, the controller causes theillumination system to illuminate the multi-well plate with an s^(th)unique illumination pattern.

FIG. 6A shows a diagram of an example arrangement of light sources 322and wells 309 illuminated according to a first illumination patternduring a first scan according to some implementations. In theillustrated implementation, the first illumination pattern is configuredor otherwise suited for a 96-well plate. As shown, while each well 309can be illuminated by multiple respective ones of the light sources 322,in the illustrated implementation, only the light sources 322 positionedat the intersections of the 1^(st), 8^(th), 15^(th) and 22^(nd) rows andthe 1^(st), 8^(th), 15^(th), 22^(nd), 29^(th) and 36^(th) columns areilluminated in the first illumination pattern during the first scan. Assuch only 24 of the light sources 322 are turned on during the firstscan in the illustrated 96-well implementation (these light sources areshown as white circles while the light sources that are not on are shownas all black). As a consequence, in the illustrated implementation, onlyone of the light sources that can illuminate a particular well actuallyilluminates the well in each scan. For example, FIG. 6A also shows anenlarged close-up view of a portion of the arrangement showing onlythose light sources 322 (49 in the illustrated implementation) that canilluminate a given well 609. As shown, only one of the possible lightsources 622 actually illuminates the well during each scan. In someimplementations, the illumination pattern changes sequentially in araster-like pattern as the scans continue throughout the series of imageacquisitions during the image acquisition phase.

For example, FIG. 6B shows the arrangement of FIG. 6A illuminatedaccording to a second illumination pattern during a second scan. In thesecond illumination pattern, only the light sources 322 positioned atthe intersections of the 1^(st), 8^(th), 15^(th) and 22^(nd) rows andthe 2^(nd), 9^(th), 16^(th), 23^(rd) and 30^(th) columns areilluminated. In such an implementation, the illumination pattern shiftsby one column to the right during each successive scan until the 7^(th)column is illuminated, at which point the illumination pattern shiftsdown to the next row and back to the first column for the next scan. Theprocess then repeats until the 7^(th) column of the 7^(th) row isreached in the n^(th) (49^(th)) scan. By way of illustration, FIG. 6Cshows the arrangement of FIG. 6A illuminated according to a 7^(th)illumination pattern during a 7^(th) scan. In the 7^(th) illuminationpattern, only the light sources 322 positioned at the intersections ofthe 1^(st), 8^(th), 15^(th) and 22^(nd) rows and the 7^(th), 14^(th),21^(st), 28^(th) and 35^(th) columns are illuminated. FIG. 6D shows thearrangement of FIG. 6A illuminated according to an 8^(th) illuminationpattern during an 8^(th) scan. In the 8^(th) illumination pattern, onlythe light sources 322 positioned at the intersections of the 2^(nd),9^(th), 16^(th) and 23^(rd) rows and the 1^(st), 8^(th), 15^(th),22^(nd), 29^(th) and 36^(th) columns are illuminated. FIG. 6E shows thearrangement of FIG. 6A illuminated according to a 42^(nd) illuminationpattern during a 42^(nd) scan. In the 42^(nd) illumination pattern, onlythe light sources 322 positioned at the intersections of the 7^(th),14^(th) and 21^(st) rows and the 1^(st), 6^(th), 15^(th), 22^(nd),29^(th) and 36^(th) columns are illuminated. Finally, FIG. 6F shows thearrangement of FIG. 6A illuminated according to a 49^(th) illuminationpattern during a 49^(th) scan. In the 49^(th) illumination pattern, onlythe light sources 322 positioned at the intersections of the 7^(th),14^(th) and 21^(st) rows and the 7^(th), 14^(th), 21^(st), 28^(th) and35^(th) columns are illuminated.

The lenses of the optical system receive (or “collect”) light scatteredby or otherwise issuing from the respective samples during each scan andfocus the received light onto the image sensors of the image sensorsystem. Although the reception and focusing of the light during eachscan is generally performed by passive elements (the lenses of theoptical system), this portion of the path of the light is still referredto as operation 508. In operation 510, each of the image sensorsreceives light focused by a corresponding lens (or set of lenses) of theoptical system acquires image data based on the focused light. Inoperation 512, the image data may be stored in one or both of a volatilememory quickly accessible by a processor of the controller or anon-volatile memory for longer term storage. As described above, theimage data represents an intensity distribution obtained during anexposure time of the scan (the image data acquired by a particular imagesensor during a particular scan is referred to as an “image frame” orsimply an “image”). In some implementations, each of the scans takesless than approximately 1 ms, enabling all n scans for an entiremulti-well plate to be completed in less than 1 second.

In some implementations, a multiplexing approach can be used to furtherdecrease the total scan time—the time required to obtain image data forn incidence angles. In one multiplexing embodiment, multiple lightsources around each well can be turned on at the same time in a uniquepattern during the capture of each raw image of each well. Using amultiplexing process, intensity data associated with each illuminationangle can be separated from the raw image captured. In this way, fewerthan n scans are required for each of the wells. An example of amultiplexing process can be found in U.S. patent application Ser. No.14/960,252 titled “MULTIPLEXED FOURIER PTYCHOGRAPHY IMAGING SYSTEMS ANDMETHODS” filed on Dec. 4, 2015, which is hereby incorporated byreference in its entirety.

In operation 514, a processor (for example, of the controller)determines whether all n of the scans have been completed. If there areremaining scans to be completed, s is incrementally updated in operation516 so that the next scan (the (s+1)^(th)) scan is then performed usingthe next (the (s+1)^(th)) illumination pattern. When all of the scansare complete, a processor (for example, of the controller) performs aparallel reconstruction process to reconstruct (or “generate”) animproved (higher) resolution image of each sample in parallel inoperation 518. During the FP reconstruction process, the n intensityimages for each sample well are iteratively combined in the Fourierdomain to generate higher-resolution image data. At each iteration, afilter is applied in the Fourier domain for a particular plane waveincidence angle, an inverse Fourier transform is applied to generate alower resolution image, the intensity of the lower resolution image isreplaced with an intensity measurement, a Fourier transform is applied,and the corresponding region in Fourier space is updated. Generally, thereconstruction process includes a phase retrieval technique that usesangular diversity to recover complex sample images. The recovery processalternates enforcement of known image data acquired in the spatialdomain and a fixed constraint in the Fourier domain. This phaseretrieval recovery can be implemented using, for example, an alternatingprojections procedure, a convex reformulation of the problem, or anynon-convex variant in-between. Instead of needing to translate a samplelaterally by mechanical means, the reconstruction process varies thespectrum constraint in the Fourier domain to expand the Fourier passbandbeyond that of a single captured image to recover a higher-resolutionsample image.

Two examples of FP reconstruction processes are discussed in detail withrespect to FIGS. 7 and 8 below. In some implementations, the controllerthen causes a display to display the reconstructed images for each ofthe wells. FIG. 7 shows a flowchart of an example FP reconstructionprocess 700 (also referred to as an “algorithm”) according to someimplementations. In some implementations, the controller 110 isconfigured perform one or more operations of the FP reconstructionprocess 700. Using this FP reconstruction process, an improvedresolution image of each of the samples in each of the sample wells isreconstructed from n low-resolution intensity distribution measurementsobtained for each sample, I_(lm) (k^(i) _(x), k_(y) ^(i)) (indexed bytheir illumination wavevector, k_(x) ^(i), k_(y) ^(i), with i=1, 2 . . .n), such as the n raw intensity images acquired during operations 506,508 and 510 of the process 500 illustrated in and described withreference to FIG. 5.

In some implementations, the FP reconstruction process 700 begins inoperation 701 with initializing a high-resolution image solution√{square root over (I_(h))}e^(iφ) ^(h) in the spatial domain. A Fouriertransform is applied to obtain an initialized Fourier transformed imageĨ_(h). In some implementations, the initial high-resolution solution isdetermined based on the assumption that the sample is located at anout-of-focus plane z=z₀. In some other implementations, the initialsolution is determined using a random complex matrix (for both intensityand phase). In some implementations, the initial solution is determinedusing an interpolation of the low-resolution intensity measurement witha random phase. In some implementations, an example of an initialsolution uses φ=0 and uses I_(h) interpolated from any low-resolutionimage of the sample area. In some implementations, an example of aninitial solution uses a constant value. Regardless, the Fouriertransform of the initial solution can be a broad spectrum in the Fourierdomain.

In the iterative operations 710, 720, 730, 740, 750, 760 and 770described below, the high-resolution image of each sample isreconstructed by iteratively combining low-resolution intensitymeasurements in Fourier space. In some implementations, operations 720and 740 may be performed if the sample is out-of-focus by the amount ofz₀.

At 710, the processor performs low-pass filtering of the high-resolutionimage √{square root over (I_(h))}e^(iφ) ^(h) in the Fourier domain togenerate a low-resolution image √{square root over (I_(l))}e^(iφ) ^(l)for a particular plane wave incidence angle (θ_(x) ^(i), θ_(y) ^(i))with a wave vector (k_(x) ^(i), k_(y) ^(i)). The Fourier transform ofthe high-resolution image is Ĩ_(h) and the Fourier transform of thelow-resolution image for a particular plane wave incidence angle isĨ_(l). In the Fourier domain, the reconstruction process filters alow-pass region from the spectrum I_(h) of the high-resolution image√{square root over (I_(h))}e^(iφ) ^(h) . The low-pass region is acircular aperture with a radius of NA*k₀, where k₀ equals 2π/λ (the wavenumber in vacuum), given by the coherent transfer function of the firstobjective lens of the IRI system. In Fourier space, the location of theregion corresponds to the illumination angle during the currentiteration. For an oblique plane wave incidence with a wave vector (k_(x)^(i), k_(y) ^(i)), the region is centered about a position (−k_(x)^(i),−k_(y) ^(i)) in the Fourier domain of √{square root over(I_(h))}e^(iφ) ^(h) .

At optional operation 720, using the processor, the low-resolutionimage, √{square root over (I_(l))}e^(iφ) ^(l) is propagated in theFourier domain to the in-focus plane at z=0 to determine thelow-resolution image at the focused position: √{square root over(I_(lf))}e^(iφ) ^(lf) . In one embodiment, operation 720 is performed byFourier transforming the low-resolution image √{square root over(I_(l))}e^(iφ) ^(l) , multiplying by a phase factor in the Fourierdomain, and inverse Fourier transforming to obtain √{square root over(I_(lf))}e^(iφ) ^(lf) . In another embodiment, operation 720 isperformed by the mathematically equivalent operation of convolving thelow-resolution image √{square root over (I_(l))}e^(iφ) ^(l) with thepoint-spread-function for the defocus. In another embodiment, operation720 is performed as an optional sub-operation of operation 710 bymultiplying Ĩ_(l) by a phase factor in the Fourier domain beforeperforming the inverse Fourier transform to produce √{square root over(I_(lf))}e^(iφ) ^(lf) . Optional operation 720 generally need not beincluded if the sample is located at the in-focus plane (z=0).

At operation 730, using the processor, the computed amplitude component√{square root over (I_(lf))} of the low-resolution image at the in-focusplane, √{square root over (I_(lf))}e^(iφ) ^(lf) , is replaced with thesquare root of the low-resolution intensity measurement √{square rootover (I_(lfm))} measured by the light detector of the IRI system. Thisforms an updated low resolution target: √{square root over(I_(lfm))}e^(iφ) ^(lf) .

At optional operation 740, using the processor, the updatedlow-resolution image √{square root over (I_(lfm))}e^(iφ) ^(lf) may beback-propagated to the sample plane (z=z₀) to determine √{square rootover (I_(ls))}e^(iφ) ^(ls) . Optional operation 740 need not be includedif the sample is located at the in-focus plane, that is, where z₀=0. Inone embodiment, operation 740 is performed by taking the Fouriertransform of the updated low-resolution image √{square root over(I_(lfm))}e^(iφ) ^(lf) and multiplying in the Fourier space by a phasefactor, and then inverse Fourier transforming it. In another embodiment,operation 740 is performed by convolving the updated low-resolutionimage √{square root over (I_(lfm))}e^(iφ) ^(lf) with thepoint-spread-function of the defocus. In another embodiment, operation740 is performed as a sub-operation of operation 750 by multiplying by aphase factor after performing the Fourier transform onto the updatedtarget image.

At operation 750, using the processor, a Fourier transform is applied tothe updated target image propagated to the sample plane: √{square rootover (I_(ls))}e^(iφ) ^(ls) , and this data is updated in thecorresponding region of high-resolution solution √{square root over(I_(h))}e^(iφ) ^(h) in the Fourier space corresponding to thecorresponding to the incidence wave vector (k_(x) ^(i), k_(y) ^(i)). Atoperation 760, the processor determines whether operations 710 through760 have been completed for all n uniquely illuminated low resolutionintensity images. If operations 710 through 760 have not been completedfor all the images, operations 710 through 760 are repeated for the nextimage.

At operation 770, the processor determines whether the high-resolutionsolution has converged. In one example, the processor determines whetherthe high-resolution solution converged to a self-consistent solution. Inone case, the processor compares the previous high-resolution solutionof the previous iteration or initial guess to the presenthigh-resolution solution, and if the difference is less than a certainvalue, the solution is determined to have converged to a self-consistentsolution. If the processor determines that the solution has notconverged at operation 770, then operations 710 through 760 arerepeated. In one embodiment, operations 710 through 760 are repeatedonce. In other embodiments, operations 710 through 760 are repeatedtwice or more. If the solution has converged, the processor transformsthe converged solution in Fourier space to the spatial domain to recoverthe improved resolution image √{square root over (I_(h))}e^(iφ) ^(h) andthe FP reconstruction process ends.

FIG. 8 shows a flowchart of another example FP reconstruction process800 (also referred to as an “algorithm”) according to someimplementations. In some implementations, the controller 110 isconfigured perform one or more operations of the FP reconstructionprocess 800. Using this FP reconstruction process, an improvedresolution image of each of the samples in each of the sample wells isreconstructed from n low-resolution intensity distribution measurementsobtained for each sample, I_(lm) (k^(i) _(x), k_(y) ^(i)) (indexed bytheir illumination wavevector, k_(x) ^(i), k_(y) ^(i), with i=1, 2 . . .n), such as the n raw intensity images acquired during operations 506,508 and 510 of the process 500 illustrated in and described withreference to FIG. 5.

In this example, the FP reconstruction process includes digitalwavefront correction. The FP reconstruction process incorporates digitalwavefront compensation in the two multiplication operations 805 and 845.Specifically, operation 805 models the connection between the actualsample profile and the captured intensity data (with includesaberrations) through multiplication with a pupil function: e^(i·φ(k)^(x) ^(,k) ^(y) ⁾ by the processor. Operation 845 inverts such aconnection to achieve an aberration-free reconstructed image. Sampledefocus is essentially equivalent to introducing a defocus phase factorto the pupil plane (i.e., a defocus aberration):

$\begin{matrix}{{e^{i \cdot {\varphi{({k_{x},k_{y}})}}} = {e^{i}}^{\sqrt{{({2{\pi/\lambda}})}^{2} - k_{x}^{2} - k_{y}^{2}} \cdot z_{0}}},{{k_{x}^{2} + k_{y}^{2}} < ( {{{NA} \cdot 2}{\pi/\lambda}} )^{2}}} & ( {{Equation}\mspace{14mu} 2} )\end{matrix}$where k_(x) and k_(y) are the wavenumbers at the pupil plane, z₀ is thedefocus distance, and NA is the numerical aperture of the firstobjective.

In some implementations, the FP reconstruction process 800 begins inoperation 801 with initializing a high-resolution image solution√{square root over (I_(h))}e^(iφ) ^(h) in the spatial domain. A Fouriertransform is applied to obtain an initialized Fourier transformed imageĨ_(h). In some implementations, the initial high-resolution solution isdetermined based on the assumption that the sample is located at anout-of-focus plane z=z₀. In some other implementations, the initialsolution is determined using a random complex matrix (for both intensityand phase). In some implementations, the initial solution is determinedusing an interpolation of the low-resolution intensity measurement witha random phase. In some implementations, an example of an initialsolution uses φ=0 and uses I_(h) interpolated from any low-resolutionimage of the sample area. In some implementations, an example of aninitial solution uses a constant value. Regardless, the Fouriertransform of the initial solution can be a broad spectrum in the Fourierdomain.

In the iterative operations of 805, 810, 830, 845, 850, 860, and 870,the high-resolution image of the sample is computationally reconstructedby iteratively combining low-resolution intensity measurements inFourier space.

In operation 805, the processor multiplies by a phase factor e^(i·φ(k)^(x) ^(,k) ^(y) ⁾ in Fourier domain. In operation 810, the processorperforms low-pass filtering of the high-resolution image √{square rootover (I_(h))}e^(iφ) ^(h) in the Fourier domain to generate alow-resolution image √{square root over (I_(l))}e^(iφ) ^(l) for aparticular plane wave incidence angle (θ_(x) ^(i), θ_(y) ^(i)) with awave vector (k_(x) ^(i), k_(y) ^(i)). The Fourier transform of thehigh-resolution image is Ĩ_(h) and the Fourier transform of thelow-resolution image for a particular plane wave incidence angle isĨ_(l). In the Fourier domain, the process filters a low-pass region fromthe spectrum Ĩ_(h) of the high-resolution image √{square root over(I_(h))}e^(iφ) ^(h) . This region is a circular aperture with a radiusof NA*k₀, where k₀ equals 2π/λ, (the wave number in vacuum), given bythe coherent transfer function of the first objective lens. In Fourierspace, the location of the region corresponds to the incidence angle.For an oblique plane wave incidence with a wave vector (k_(x) ^(i),k_(y) ^(i)), the region is centered about a position (−k_(x) ^(i),−k_(y)^(i)) in the Fourier domain of √{square root over (I_(h))}e^(iφ) ^(h) .

In operation 830, using the processor, the computed amplitude component√{square root over (I_(lf))} of the low-resolution image at the in-focusplane, √{square root over (I_(lf))}e^(iφ) ^(lf) , is replaced with thesquare root of the low-resolution intensity measurement √{square rootover (I_(lfm))} measured by the light detector of the IRI system. Thisforms an updated low resolution target: √{square root over(I_(lfm))}e^(iφ) ^(lf) . In operation 845, the processor multiplies byan inverse phase factor e^(i·φ(k) ^(x) ^(,k) ^(y) ⁾ in Fourier domain.At operation 850, a Fourier transform is applied to the updated targetimage propagated to the sample plane: √{square root over (I_(ls))}e^(iφ)^(ls) , and this data is updated in the corresponding region ofhigh-resolution solution √{square root over (I_(h))}e^(iφ) ^(h) in theFourier space corresponding to the corresponding to the incidence wavevector (k_(x) ^(i), k_(y) ^(i)). In operation 860, the processordetermines whether operations 805 through 850 have been completed forall n uniquely illuminated low resolution intensity images. Ifoperations 805 through 850 have not been completed for all for all nuniquely illuminated low resolution intensity images, operations 805through 850 are repeated for the next image.

In operation 870, the processor determines whether the high-resolutionsolution has converged. In one example, the processor determines whetherthe high-resolution solution has converged to a self-consistentsolution. In one case, the processor compares the previoushigh-resolution solution of the previous iteration or initial guess tothe present high-resolution solution, and if the difference is less thana certain value, the solution has converged to a self-consistentsolution. If processor determines that the solution has not converged,then operations 805 through 870 are repeated. In one embodiment,operations 805 through 870 are repeated once. In other embodiments,operations 805 through 870 are repeated twice or more. If the solutionhas converged, the processor transforms the converged solution inFourier space to the spatial domain to recover the high-resolution image√{square root over (I_(h))}e^(iφ) ^(h) and the FP reconstruction processends.

Additional details of example FP reconstruction processes can be foundin Zheng, Guoan, Horstmeyer, Roarke, and Yang, Changhuei, “Wide-field,high-resolution Fourier ptychographic microscopy,” Nature Photonics vol.7, pp. 739-745 (2013) and in U.S. patent application Ser. No.14/065,280, titled “Fourier Ptychographic Imaging Systems, Devices, andMethods” and filed on Oct. 28, 2013, both of which are herebyincorporated by reference herein in their entireties and for allpurposes.

FIG. 9 shows a flowchart depicting operations of an example calibrationprocess 900 for determining the angles of incidence for each of thewells of a multi-well plate. For example, the calibration process 900can be performed during the initialization process in block 504 of theprocess 500 described with reference to FIG. 5. In block 921, thecontroller causes the illumination system to illuminate a central lightelement (for example, a respective one of the light sources 322)corresponding to at least one of the wells selected for the calibrationprocess 900, and in some implementations, a plurality or even all of thewells. In some implementations, for example, the controller causes theillumination system to illuminate a central light element for only thosewells in the four corners of the multi-well plate or only those wells inone or more particular columns and particular rows. In one embodiment,the controller determines the central light element by turning on lightelements sequentially and capturing an image for each light elementillumination. The central light element is determined based on thehighest intensity image captured during the illumination by the multiplelight elements.

In block 922, the controller causes each of the respective image sensorsselected for the calibration process 900 to capture a vignettemonochromic image during illumination by the central light element. Insome implementations, the image is then converted to black and white. Ifthere is a misalignment between the light element and the image sensor,the center of the image is shifted from the center of the image sensor.In block 923, the center of the image is determined. In block 924, theshift of the center of the image is measured along an x-axis direction(x-shift) and along a y-axis direction (y-shift). At operation 925, thedisplacement of the central light element is determined based on thex-shift and y-shift of the image using a lookup table or plot. Thelookup table/plot provides different displacements of the central lightelement associated with different values of x-shift and y-shift. Oncethe displacement of the central light element is determined from thelookup table/plot, the illumination angles associated with the lightelements in the variable illumination source can be determined based onthe geometry of the variable illumination source. In block 926, precisevalues of the n illumination angles associated with the FP illuminationare determined using the displacement of the central light element.

FIG. 10A shows a vignette monochromic image captured during illuminationby a central LED of an LED matrix according to one example. FIG. 10B isa converted black and white version of the image of FIG. 10A. In thisexample, the center 1022 of the black and white image is located at thesame location as the center 1023 of the image sensor and the LEDposition is well aligned with the imaging sensor of the CMOS camera.FIG. 10C shows an image captured during illumination by a center LED ofan LED matrix according to another example. In the example shown in FIG.10C, there is a misalignment between the central LED and the imagesensor. More specifically, there is a shift between the center 1032 ofthe image and the center 1023 of the image sensor. Even morespecifically, there is a shift in the x direction (pixel shift X) and ashift in they direction (pixel shift Y). FIG. 11 shows a lookup plot ofLED displacement associated with x-shift and y-shift of the center 1032of the image with respect to the center 1023 of the image sensor,according to an embodiment. In this example, the lookup table was madeby moving the LED matrix relative to the image sensor by known amountsand determining different shifts of the center of the image associatedwith the LED displacement.

FIG. 12 shows a flowchart illustrating an example fluorescence imagingprocess 1200 for imaging a multi-well plate according to someimplementations. For example, the process 1200 also can be performedusing the systems, devices and arrangements described above with respectto FIGS. 1-4. In some implementations, the controller 110 is configuredperform one or more operations of the fluorescence imaging process 1200.In some implementations, the fluorescence imaging process 1200 begins inoperation 1202 with loading a multi-well plate into the imaging system.In some other implementations, the fluorescence imaging process 1200 canbe performed automatically immediately (or shortly) after the FP imagingprocess 500 ends. In some other implementations, the fluorescenceimaging process 1200 can begin between operations 502 and 506.

In operation 1204, the controller initializes the illumination systemand the image sensor system. Initializing the illumination system inoperation 1204 can include retrieving illumination information (such asthe wavelength(s) of the excitation signals) from a non-volatile memoryand loading the retrieved illumination information into a volatilememory for subsequent use in performing the imaging. Initializing theimage sensor system in operation 1204 can include powering on orotherwise preparing the image sensors to receive light and to generateimage data.

In operation 1206, the controller causes the illumination system toilluminate the multi-well plate with the excitation light. As describedabove, in operation 1206 all of the light sources 322 can be turned onsimultaneously at a particular wavelength or within a particular rangeof wavelengths. Fluorophore in the sample are activated by theexcitation light and emit light (emissions) of another range ofwavelengths (e.g., blue, green or red light).

The lenses of the optical system receive (or “collect”) light emitted bythe respective samples and focus the received light onto the imagesensors of the image sensor system. An optical filter filters the lightsuch that only the light emitted by the fluorophore is propagated to theimage sensors. Although the reception, focusing and filtering of thelight is generally performed by passive elements (the lenses of theoptical system and a color filter), this portion of the path of thelight is still referred to as operation 1208 and 1210. In operation1212, each of the image sensors receives light focused by acorresponding lens (or set of lenses) of the optical system and acquiresfluorescence image data based on the focused light. In operation 1214,the image data may be stored in one or both of a volatile memory quicklyaccessible by a processor of the controller or a non-volatile memory forlonger term storage. As described above, the image data represents anintensity distribution obtained during an exposure time.

In some implementations, in operation 1216, the processor determineswhether a desired intensity has been achieved. If a desired intensityhas not been achieved, operations 1206 through 1214 can be repeatedmultiple times and the resultant acquired fluorescence image data foreach of the sample wells can be added or otherwise combined together inoperation 1218 to obtain a desired intensity image for each of thesample wells.

In some implementations, for multi-band, multichannel embodiments,operations 1206 through 1214 can be repeated multiple times for each ofmultiple regions of the light spectrum (referred to as “bands”), and insome implementations, using different filters.

In some implementations, the controller generates a combinedfluorescence and high resolution bright-field image of the sample byoverlaying a fluorescence image generated by the fluorescence imagingprocess 1200 and a high resolution bright-field image generated by theFP imaging process 500. In another aspect, the processor generates acombined fluorescence and low resolution bright-field image of thesample by overlaying a fluorescence image generated by the fluorescenceimaging process and a low resolution bright-field image captured duringthe acquisition process of the FP imaging process. In another aspect,the processor generates a high resolution phase image of the samplebased on phase data in the FP imaging process.

In some implementations, the imaging systems described herein also canimplement time-lapse imaging or other long term imaging. For example,the imaging processes 500 and 1200 can repeat at intervals such as, forexample, one hour intervals, two hour intervals, one day intervals, etc.The imaging method can continue repeating each imaging run at intervalsfor a set period of time (e.g., one week, two weeks, one month, twomonths, etc.) or can run until an operator stops the imaging method. Insome implementations, while this long term imaging continues, theimaging system can be located within an incubator.

Various modifications to the implementations described in thisdisclosure may be readily apparent to those skilled in the art, and thegeneric principles defined herein may be applied to otherimplementations without departing from the spirit or scope of thisdisclosure. Thus, the claims are not intended to be limited to theimplementations shown herein, but are to be accorded the widest scopeconsistent with this disclosure, the principles and the novel featuresdisclosed herein.

Additionally, particular features that are described in thisspecification in the context of separate implementations also can beimplemented in combination in a single implementation. Conversely,various features that are described in the context of a singleimplementation also can be implemented in multiple implementationsseparately or in any suitable subcombination. Moreover, althoughfeatures may be described above as acting in particular combinations andeven initially claimed as such, one or more features from a claimedcombination can in some cases be excised from the combination, and theclaimed combination may be directed to a subcombination or variation ofa subcombination.

Similarly, while various operations (also referred to herein as“blocks”) are depicted in the drawings in a particular order, thisshould not be understood as requiring that such operations be performedin the particular order shown or in sequential order, or that allillustrated operations be performed, to achieve desirable results.Further, the drawings may schematically depict one more exampleprocesses in the form of a flow diagram. However, other operations thatare not depicted can be incorporated in the example processes that areschematically illustrated. For example, one or more additionaloperations can be performed before, after, simultaneously, or betweenany of the illustrated operations. Moreover, the separation of varioussystem components in the implementations described above should not beunderstood as requiring such separation in all implementations.Additionally, other implementations are within the scope of thefollowing claims. In some cases, the actions recited in the claims canbe performed in a different order and still achieve desirable results.

It will also be understood by persons having ordinary skill in the artthat various functions, operations, processes, modules or componentsthat described above can be implemented in the form of control logicusing computer software in a modular or integrated manner. Based on thedisclosure and teachings provided herein, a person of ordinary skill inthe art will know and appreciate other ways and/or methods to implementthe present invention using hardware and a combination of hardware andsoftware.

Any of the software components or functions described in thisapplication, may be implemented as software code to be executed by aprocessor using any suitable computer language such as, for example,Java, C++ or Perl using, for example, conventional or object-orientedtechniques. The software code may be stored as a series of instructions,or commands on a CRM, such as a random access memory (RAM), a read onlymemory (ROM), a magnetic medium such as a hard-drive or a floppy disk,or an optical medium such as a CD-ROM. Any such CRM may reside on orwithin a single computational apparatus, and may be present on or withindifferent computational apparatuses within a system or network.

What is claimed is:
 1. A system for array-level Fourier ptychographicimaging, the system comprising: a plurality of optical arrangements,each optical arrangement having one or more lenses configured to receivelight from a corresponding sample well of an array of sample wells in amulti-well plate received into the system during operation; a pluralityof image sensors configured to receive light from the plurality ofoptical arrangements and acquire image data based on the light received;an illumination system configured to illuminate the array of samplewells in parallel such that each sample well is illuminated sequentiallywith light at different angles of incidence; and a processor configuredto perform a Fourier ptychographic operation to generate a reconstructedimage for each of the sample wells based on the image data acquired forthe respective sample well from light provided at the different anglesof incidence by the illumination system.
 2. The system of claim 1,wherein each optical arrangement comprises a first lens and a secondlens.
 3. The system of claim 2, wherein each optical arrangement furthercomprises a third lens.
 4. The system of claim 1, wherein theillumination system comprises: an array of light elements configured toilluminate the array of sample wells in parallel; and at least one ormore excitation light sources, each excitation light source configuredto illuminate one of the sample wells, wherein each excitation lightsource is configured to provide excitation light with power higher thanthe illumination from the array of light elements.
 5. The system ofclaim 4, wherein the processor is further configured to generate afluorescence image for each of the sample wells illuminated byexcitation light from the one or more excitation light sources.
 6. Thesystem of claim 4, wherein each optical arrangement further comprises afilter configured to filter excitation light, the filter located betweenthe multi-well plate and the array of image sensors.
 7. The system ofclaim 1, wherein the plurality of optical arrangements comprises atleast one lens array.
 8. The system of claim 1, wherein the multi-wellplate has one of 6 wells, 12 wells, 96 wells, 384 wells, 1536 wells,3456 wells, or 9600 wells.
 9. The system of claim 1, wherein theillumination system includes an array of light elements configured toilluminate the array of sample wells in parallel.
 10. The system ofclaim 9, wherein the array of light elements is a light emitted diodematrix.
 11. The system of claim 9, wherein the array of light elementsis configured to sequentially activate light elements to generate asequence of different illumination patterns.
 12. The system of claim 9,wherein the array of light elements is configured such that eachactivated light element illuminates a plurality of sample wells at eachexposure time.
 13. The system of claim 9, wherein the array of lightelements is configured such that each activated light elementilluminates a single sample well at each exposure time.
 14. The systemof claim 1, wherein the Fourier ptychographic operation includesaberration correction for correcting for any aberrations in the system.15. A system for imaging an array of sample wells, the systemcomprising: at least one lens array, each lens configured to receivelight from a corresponding sample well of the array of sample wells; aplurality of image sensors configured to receive light from the at leastone array of lenses and acquire image data based on the received light;an array of light elements configured to illuminate the array of samplewells in parallel with illumination such that each sample well isprovided sequentially with illumination at different angles ofincidence; at least one excitation light source configured to illuminateat least one sample well of the array of sample wells, wherein the atleast one excitation light source provides illumination at a differenttime than the array of light elements; and a processor configured to:perform a Fourier ptychographic operation to generate a reconstructedimage for each sample well based on the image data acquired for therespective sample well from the sequential illumination with light atdifferent angles of incidence, and generate, for each of the at leastone sample well, a fluorescence image based on the image data acquiredfor the respective sample well from the excitation light.
 16. The systemof claim 15, further comprising an optical filter configured to filterexcitation light, the optical filter located between the array of samplewells and the plurality of image sensors.
 17. The system of claim 16,wherein the at least one excitation light source is side-mounted. 18.The system of claim 15, wherein the at least one excitation light sourceprovides excitation light with power higher than power of theillumination from the array of light elements.
 19. The system of claim15, wherein the array of sample wells is part of a multi-well plate. 20.The system of claim 19, wherein the multi-well plate has one of 6 wells,12 wells, 96 wells, 384 wells, 1536 wells, 3456 wells, or 9600 wells.21. The system of claim 15, wherein the array of light elements is alight emitted diode matrix.
 22. The system of claim 15, wherein thearray of light elements is configured to sequentially activate differentlight elements of the array of light elements to generate a sequence ofdifferent illumination patterns.